Method for Storing Gaseous Hydrogen Through Producing Methanoate (Formate)

ABSTRACT

The present invention relates to a method for storing gaseous hydrogen, comprising the steps of producing methanoate (formate) through contacting gaseous hydrogen with carbon dioxide in the presence of a hydrogen dependent carbondioxide reductase (HDCR), and thereby storing of said gaseous hydrogen. The HDCR and/or its complex is preferably derived from  Acetobacterium woodii.

The present invention relates to a method for storing gaseous hydrogen,comprising the steps of producing methanoate (formate) throughcontacting gaseous hydrogen with carbon dioxide in the presence of ahydrogen dependent carbon dioxide reductase (HDCR), and thereby storingof said gaseous hydrogen. The HDCR and/or its complex is preferablyderived from Acetobacterium woodii.

BACKGROUND OF THE INVENTION

One promising alternative to fossil fuels is hydrogen. Through itsreaction with oxygen, hydrogen releases energy explosively in heatengines or quietly in fuel cells to produce water as its only byproduct.Hydrogen is abundant and generously distributed throughout the worldwithout regard for national boundaries. Storing hydrogen in ahigh-energy-density form that flexibly links its production and eventualuse is a key element of the hydrogen economy.

Boddien et al. (in: CO₂-“Neutral” Hydrogen Storage Based on Bicarbonatesand Formates. Angew. Chem. Int. Ed., 2011 50: 6411-6414) describe aruthenium catalyst generated in situ that facilitates the selectivehydrogenation of bicarbonates and carbonates, as well as CO₂ and base,to give formates and also the selective dehydrogenation of formates backto bicarbonates. The two reactions can be coupled, leading to areversible hydrogen-storage system.

KR 2004/0009875 describes an electrochemical preparation method offormic acid using carbon dioxide, thereby simultaneously carrying outreduction of carbon dioxide and conversion of carbon dioxide into usefulorganic matters. The method comprises electrochemical reduction ofcarbon dioxide using formic acid dehydrogenase or formic aciddehydrogenase-producing anaerobic bacteria and an electron carrier inwhich reversible oxidation/reduction is occurred at electric potentialof −400 to −600 mV, wherein the concentration of the electron carrier is5 to 15 mM; the anaerobic bacteria are selected from Clostridiumthermoaceticum, Clostridium thermoauthotrophicum, Acetobacterium woodii,Acetogenium kivui, Clostridium aceticum, Clostridium ljungdahlii,Eubacterium limosum or a mixture thereof; the electron carrier isselected from methylviologen, N,N,-diethyl-4,4-bipyridyl,N,N-diisopropylyl-4,4-bipyridyl, 4,4-bipyridyl or a mixture thereof; thereduction temperature is 20 to 70° C.; and the reduction pH is 6.0 to7.0.

WO 2011/087380 describes methods for improving the efficiency of carboncapture in microbial fermentation of a gaseous substrate comprising COand/or H₂; said method comprising applying an electrical potentialacross the fermentation. It further relates to improving the efficiencyof carbon capture in the microbial fermentation of gaseous substratecomprising CO and/or H₂ to produce alcohol(s) and/or acid (s).

Catalytic processes may be used to convert gases consisting primarily ofCO and/or CO and hydrogen (H₂) into a variety of fuels and chemicals.Microorganisms may also be used to convert these gases into fuels andchemicals. These biological processes, although generally slower thanchemical reactions, have several advantages over catalytic processes,including higher specificity, higher yields, lower energy costs andgreater resistance to poisoning.

The ability of microorganisms to grow on CO as a sole carbon source wasfirst discovered in 1903. This was later determined to be a property oforganisms that use the acetyl coenzyme A (acetyl CoA) biochemicalpathway of autotrophic growth (also known as the Wood-Ljungdahl pathwayand the carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS)pathway). A large number of anaerobic organisms includingcarboxydotrophic, photosynthetic, methanogenic and acetogenic organismshave been shown to metabolize CO to various end products, namely CO₂,H₂, methane, n-butanol, acetate and ethanol. While using CO as the solecarbon source, all such organisms produce at least two of these endproducts. The Wood-Ljungdahl pathway of anaerobic CO₂ fixation withhydrogen as reductant is considered a candidate for the firstlife-sustaining pathway on earth because it combines carbon dioxidefixation with the synthesis of ATP via a chemiosmotic mechanism.

Schuchmann et al. (A bacterial electron-bifurcating hydrogenase. J BiolChem. 2012 Sep. 7; 287(37):31165-71) describe a multimeric[FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD)catalyzing hydrogen-based ferredoxin reduction. Apparently, themultimeric hydrogenase of A. woodii is a soluble energy-convertinghydrogenase that uses electron bifurcation to drive the endergonicferredoxin reduction by coupling it to the exergonic NAD⁺ reduction.

Schiel-Bengelsdorf, and Dürre (in:Pathway engineering and syntheticbiology using acetogens, FEBS Letters, 2012, 586, 15, 2191) describeacetogenic anaerobic bacteria that synthesize acetyl-CoA from CO₂ or CO.Their autotrophic mode of metabolism offers the biotechnological chanceto combine use of abundantly available substrates with reduction ofgreenhouse gases. Several companies have already established pilot anddemonstration plants for converting waste gases into ethanol, animportant biofuel and a natural product of many acetogens. RecombinantDNA approaches now opened the door to construct acetogens, synthesizingimportant industrial bulk chemicals and bio fuels such as acetone andbutanol. Thus, novel microbial production platforms are available thatno longer compete with nutritional feedstocks.

WO 2011/028137 describes a bioreactor system for fermentation of agaseous substrate comprising CO and optionally H₂, or CO₂ and H2, to oneor more products, including acid(s) and/or alcohol(s).

U.S. Pat. No. 7,803,589 describes an Escherichia coli microorganism,comprising a genetic modification, wherein said genetic modificationcomprises transformation of said microorganism with exogenous bacterialnucleic acid molecules encoding the proteins cobalamidecorrinoid/iron-sulfur protein, methyltransferase, carbon monoxidedehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfidereductase and hydrogenase, whereby expression of said proteins increasesthe efficiency of producing acetyl-CoA from CO₂, CO or H₂, or acombination thereof.

Poehlein et al. (An ancient pathway combining carbon dioxide fixationwith the generation and utilization of a sodium ion gradient for ATPsynthesis. PLoS One. 2012;7(3): e33439. doi:10.1371/joumal.pone.0033439)describes that the synthesis of acetate from carbon dioxide andmolecular hydrogen is considered to be the first carbon assimilationpathway on earth. It combines carbon dioxide fixation into acetyl-CoAwith the production of ATP via an energized cell membrane. How thepathway is coupled with the net synthesis of ATP has been an enigma. Theanaerobic, acetogenic bacterium Acetobacterium woodii uses an ancientversion of this pathway without cytochromes and quinones. It generates asodium ion potential across the cell membrane by the sodium-motiveferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodiisolves the enigma: it uncovers Rnf as the only ion-motive enzyme coupledto the pathway and unravels a metabolism designed to produce reducedferredoxin and overcome energetic barriers by virtue ofelectron-bifurcating, soluble enzymes.

As mentioned above, hydrogen is one of the most discussed future energysources. Methods for producing hydrogen are well known, but storage andtransport of the gas is an unsolved problem. It is therefore an objectof the present invention, to provide new and effective methods in orderto provide for new ways to store hydrogen, in particular directly fromthe gaseous phase. Other objects and advantages will become apparent tothe person of skill upon studying the following description and theexamples of the invention.

According to a first aspect thereof, the object of the present inventionis solved by providing a method for storing gaseous hydrogen, comprisingthe steps of producing methanoate (formate) through contacting gaseoushydrogen with carbon dioxide in the presence of a hydrogen dependentcarbon dioxide reductase (HDCR), and thereby storing of said gaseoushydrogen.

The present invention is based on the surprising finding that the HDCRenzyme, and preferably a respective enzyme complex, has been found toconvert gaseous H₂+CO₂ directly into formate in the reactionH₂+CO₂→HCOOH. The present biological system functions at normal, such asambient, pressure and temperature, preferably at standard ambienttemperature and pressure or at between about 20° C. to about 40° C. andnormal pressure. The method furthermore has a high conversion rate,compared with known chemical catalysts. Also, preferably no additionalenergy has to be provided.

Since the reaction takes place closely to the thermodynamic equilibrium,in the reverse reaction, hydrogen can be readily released from theformate.

In contrast to the H₂ to be converted, the CO₂ can be provided in themethod both in gaseous and/or solid from. Preferred is a method of thepresent invention, wherein the CO₂ is provided in the form of hydrogencarbonate (HCO3⁻) (see also FIG. 3).

Preferred is a method according to the present invention, wherein themethod does not involve electrochemical reduction, in particular ofcarbon dioxide. No electric energy has to be provided, and in particularno means for providing an electrical potential to a bioreactor asinvolved.

Preferred is a method according to the present invention, wherein saidHDCR is selected from a bacterial enzyme, such as, for example FdhF1(Acc. No. YP_005268500, SEQ ID No.1) or FdhF2 (Acc. No. YP_005268502,SEQ ID No.2) of Acetobacterium woodii. Also preferred are formatedehydrogenase enzymes that are at least 65% identical to the FdhF1and/or FdhF2 enzyme, more preferably at least 70%, even more preferredat least 80%, and most preferred at least 90% identical to the FdhF1and/or FdhF2 enzyme on the amino acid level. Preferred examples areselected from the formate dehydrogenase-H of Clostridium difficile 630(Acc. No. YP_001089834.2), the formate dehydrogenase h of Clostridiumdifficile CD196 (Acc. No. YP_003216147.1), the formate dehydrogenase ofClostridium sp. DL-VIII (Acc. No. WP_009172363.1), the formatedehydrogenase of Clostridium arbusti (Acc. No. WP_010238540.1), theformate dehydrogenase of Clostridium ragsdalei (Acc. No. gb|AEI90724.1),the formate dehydrogenase H of Paenibacillus polymyxa E681 (Acc. No.YP_003871035.1), the formate dehydrogenase-H of Clostridium difficile630 (Acc. No. YP_001089834.2), the formate dehydrogenase h ofClostridium difficile CD196 (Acc. No. YP_003216147.1), the formatedehydrogenase H of Treponema primitia ZAS-2 (Acc. No. ADJ19611.1), theformate dehydrogenase H of Clostridium carboxidivorans P7 (Acc. No.ADO12080.1), and the formate dehydrogenase I of Clostridium ragsdalei(Acc. No. gb|AEI90722.1), and mixtures thereof. All these proteins shallbe understood as “homologs” of the proteins of Acetobacterium woodii asdescribed herein.

Further preferred is a method according to the present invention,wherein said HDCR is part of an enzyme complex, for example with aformate dehydrogenase accessory protein, such as, for example, FdhD ofAcetobacterium woodii, an electron transfer protein, such as, forexample, HycB1 or HycB2 of Acetobacterium woodii, and a subunitharboring the active site characteristic of an [FeFe]-hydrogenase, suchas, for example, HydA2 of Acetobacterium woodii. Also preferred areformate dehydrogenase accessory proteins and/or electron transferproteins and/or [FeFe]-hydrogenase proteins, that are at least 65%identical to the HydA2, FdhD, HycB1 and/or HycB2 enzyme, more preferablyat least 70%, even more preferred at least 80%, and most preferred atleast 90% identical to the HydA2, FdhD, HycB1 and/or HycB2 enzyme on theamino acid level, and show an electron transfer activity, formatedehydrogenase accessory protein activity, or [FeFe]-hydrogenaseactivity. Also these proteins shall be understood as “homologs” of theproteins of Acetobacterium woodii as described herein.

Particularly preferred is a method according to the present invention,wherein said HDCR is part of the enzyme complex comprising FdhF1/2,HycB1/2/3, and HydA2. Thereby, FdhF reduces CO₂ to formate, theelectrons are provided by HydA2, the subunit of the H₂-oxidation. Morepreferably the HDCR is a protein complex composed of the subunitsFdhF1/FdhF2, HycB1/HycB2, HydA2 and HycB3. Most preferably the HDCR isselected from one of the complexes comprising FdhF1, HycB1, HydA2 andHycB3, or the complex comprising FdhF2, HycB2, HydA2 and HycB3

Further preferred is a method according to the present invention,wherein said method further comprises an inhibition of the cellularmetabolism to further metabolize formate, such as an Na depletion, forexample using sodium ionophores. When the metabolism of the cell isinhibited (and/or impaired), the formate as produced can no longer reactfurther, and is advantageously produced as the final product. For theinhibition of the energy metabolism, all substances can be used that areknown to the person of skill, and examples are selected from all ATPaseinhibitors, such as DCCD (dicyclohexylcarbodiimide), heavy metals suchas silver ions, copper ions, etc., all decoupling agents of the membranepotential, such as protonophores such as TCS(3,3′,4′,54tetrachlorosalicylanilide), K-ionophores, such asvalinomycine, propyl iodide as inhibitor of cobalt dependent reactions,phosphate starvation, which slows down ATP-synthesis, and tensides orsubstances that destroy the integrity of the membrane of the cell. It isimportant, that an enzyme and/or step of the energy metabolism isblocked, since this leads to an accumulation of the intermediateproduct. Since the HDCR is independent from the energy metabolism anddoes not require external electron carriers or energy, the process offormate formation can continue. This phenomenon can of course be appliedboth to reactions in whole cells, as well as in in vitro-reactions. Theinventors have furthermore surprisingly found that the synthesis ofacetyl-CoA can be stopped at formate, if Na is depleted. The system (forexample bacteria) then nearly exclusively produces formate, which isused for hydrogen storage. Depletion can be achieved by usingsodium-free buffers and/or media, and/or by using sodium-ionophores,such as, for example, Monensin, Gramicidin A, or the commerciallyavailable ETH 2120(N,N,N′,N′-Tetracyclohexyl-1,2-phenylenedioxydiacetamide,Selectophore™), or the like.

In another aspect of the present invention the present invention thus isbased on the surprising finding that the inhibition of the cellularmetabolism to further metabolize formate, such as by Na depletion (forexample using sodium ionophores) can be advantageously used to produceformate. In this embodiment, the Na depletion leads to an accumulationof formate based on the effective blocking of the production ofdownstream products from the formate. The present invention thus furtherrelates to a method for producing methanoate (formate) comprisingcontacting carbon dioxide in the presence of a hydrogen dependent carbondioxide reductase (HDCR) under conditions that inhibit the cellularmetabolism to further metabolize formate, such as, for example, under Nadepletion, at an electric potential of −300 to −600 mV (e.g. usingelectrodes) and/or in the presence of an electron carrier. Theconcentration of the electron carrier can be from 5 to 15 mM, and theelectron carrier can be selected from methylviologen,N,N,-diethyl-4,4-bipyridyl, N,N-diisopropylyl-4,4-bipyridyl,4,4-bipyridyl or a mixture thereof. Preferably, said HDCR is selectedfrom a bacterial enzyme, such as, for example FdhF1 or FdhF2 ofAcetobacterium woodii. More preferably the HDCR is a protein complexcomposed of at least one of the subunits FdhF1/FdhF2, HycB1/HycB2, HydA2and HycB3. Most preferably the HDCR is selected from one of thecomplexes comprising FdhF1, HycB1, HydA2 and HycB3, or the complexcomprising FdhF2, HycB2, HydA2 and HycB3. Further preferably, saidmethod is performed under standard ambient temperature and pressure, orat between about 20° C. to about 40° C. and normal pressure. Otherpreferred embodiments of this method are analogous as described hereinfor the first aspect of the present invention.

Yet another aspect of the present invention then relates to a methodaccording to the present invention, further comprising the step ofconverting carbon monoxide into carbon dioxide using a CO dehydrogenase,such as, for example a bacterial CO dehydrogenase, such as, for exampleAcsA of Acetobacterium woodii, and a ferredoxin. Also preferred are COdehydrogenase enzymes that are at least 65% identical to the AcsAenzyme, more preferably at least 70%, even more preferred at least 80%,and most preferred at least 90% identical to the AcsA enzyme on theamino acid level, and show a CO dehydrogenase activity. All theseproteins shall be understood as “homologs” of the proteins ofAcetobacterium woodii as described herein.

In this aspect of the present invention, it was furthermore surprisinglyfound that the enzyme hydrogen dependent carbon dioxide reductase (HDCR)can also use carbon monoxide (via ferredoxin) as electron donor for theCO₂-reduction to formate. Thus, this enables the advantageous use ofsynthesis gas (for example as feed-stock) for the method. Of course,this aspect of the invention also can be performed under the conditionsand using the complex and enzymes as described above for the “direct”CO₂-use. Furthermore, this aspect of the method of the invention can beused to remove CO from gaseous phases, and thus can constitute a methodfor decontaminating CO-contaminated (or polluted) gases.

Yet another aspect of the present invention thus relates to a method fordecontaminating CO-contaminated or polluted gases, comprising performinga method according to the invention as above using said CO-contaminatedor polluted gas as a substrate. Preferably, said CO-contaminated orpolluted gas is synthesis gas.

In the methods according to the present invention, both purified (orpartially purified) enzyme(s) as well as bacterial cells can be used.Thus, methods according to the present invention can be performed invitro, in vivo and/or in culture, for example in imidazole buffer (seebelow).

Most preferred is a method according to the present invention, which isperformed in a bioreactor in a continuous operation or in batches.Respective methods and devices are known to the person of skill anddescribed (for example, in Demler and Weuster-Botz; Reaction engineeringanalysis of hydrogenotrophic production of acetic acid by Acetobacteriumwoodii. Biotechnol Bioeng. 2011 Feb; 108(2):470-4).

Yet another aspect of the present invention thus relates to arecombinant bacterial organism comprising a genetic modification,wherein said genetic modification comprises transformation of saidmicroorganism with exogenous bacterial nucleic acid molecules encodingthe proteins FdhF1 and/or FdhF2, FdhD, HycB1 and/or HycB2, HydA2, andoptionally HycB3 or AcsA of Acetobacterium woodii, or homologs thereofas described herein, whereby expression of said proteins increases theefficiency of producing format from CO₂, and/or CO and H₂. Morepreferably the nucleic acids encode at least one of the HDCR subunitsFdhF1/FdhF2, HycB1/HycB2, HydA2 and HycB3. Most preferably the nucleicacids encode the proteins FdhF1, HycB1, HydA2 and HycB3, or the proteinsFdhF2, HycB2, HydA2 and HycB3.

Further preferred is a method according to the present invention,further comprising the (recombinant) expression of the genes ofhydrogenase maturation as well as for a cofactor biosynthesis of theformate-dehydrogenase (e.g. described in Kuchenreuther J. M.,Grady-Smith C. S., Bingham A. S., George S. J., Cramer S. P., et al.(2010) High-Yield Expression of Heterologous [FeFe] Hydrogenases inEscherichia coli. PLoS ONE 5(11): e15491.doi:10.1371/journal.pone.0015491).

Preferred is the use of the recombinant bacterial organism according tothe present invention in a method according to the present invention asdescribed herein.

Yet another aspect of the present invention relates to the use of ahydrogen dependent carbon dioxide reductase (HDCR), for example abacterial enzyme, such as, for example FdhF1 or FdhF2 of Acetobacteriumwoodii or homologs thereof in a method according to the presentinvention as described herein. Preferred is a use, wherein said HDCR ispart of an enzyme complex, for example with a formate dehydrogenaseaccessory protein, such as, for example, FdhD of Acetobacterium woodii,an electron transfer protein, such as, for example, HycB1 or HycB2 ofAcetobacterium woodii, and a subunit harboring the active sitecharacteristic of an [FeFe]-hydrogenase, such as, for example, HydA2 ofAcetobacterium woodii, or homologs thereof. Further preferred is a useaccording to the present invention, wherein said complex furthercomprises a CO dehydrogenase, such as, for example a bacterial COdehydrogenase, such as, for example AcsA of Acetobacterium woodii, and aferredoxin, or homologs thereof. More preferably the HDCR is a proteincomplex composed of at least one of the subunits FdhF1/FdhF2,HycB1/HycB2, HydA2 and HycB3. Most preferably the HDCR is selected fromone of the complexes comprising FdhF1, HycB1, HydA2 and HycB3, or thecomplex comprising FdhF2, HycB2, HydA2 and HycB3.

The following figures, sequences, and examples merely serve toillustrate the invention and should not be construed to restrict thescope of the invention to the particular embodiments of the inventiondescribed in the examples. For the purposes of the present invention,all references as cited in the text are hereby incorporated in theirentireties.

FIG. 1 shows the productions of formate using whole cell catalysis. Cellsuspensions of A. woodii (1 mg/ml) were incubated using a gas phase of0.8×10⁵ Pa H₂ and 0.2×10⁵ Pa CO₂ (A). Adding the Na⁺ ionophore ETH2120(30 μM) gives rise to the production of up to 8 mM formate and theproduction of acetate stopped (B).

FIG. 2 shows the production of formate using HCO₃ ⁻ or CO₂ as substrate.Cell suspensions of A. woodii (1 mg/ml) were incubated using a gas phaseof 0.8×10⁵ Pa H₂ and 0.2×10⁵ Pa CO₂ or 1×10⁵ Pa H₂ with 300 mM KHCO₃.

FIG. 3 shows the relationship of final formate concentration to initialHCO₃ ⁻. Cell suspensions of A. woodii (1 mg/ml) were incubated withincreasing amounts of initial HCO₃ ⁻ and a gas phase of 1×10⁵ Pa H₂.

Sequence ID NOs. 1 to 8 show the amino acid sequences of the enzymesFdhF1, HycB1, FdhF2, HycB2, FdhD, HycB3, HydA2, and AcsA of A. woodii,respectively.

EXAMPLES

Measurements with the Isolated HCDR

For the purification of HCDR A. woodii (DSM 1030) was grown at 30° C.under anaerobic conditions in 20-1-liter flasks using 20 mM fructose toan OD₆₀₀ of ˜2.5. All buffers used for preparation of cell extracts andpurification contained 2 mM DTE and 4 μM resazurin. All purificationsteps were performed under strictly anaerobic conditions at roomtemperature in an anaerobic chamber filled with 100% N₂ and 2-5% H₂. Thecell free extract was prepared as descirbed previously (Schuchmann etal., J Biol Chem. 2012 Sep. 7; 287(37):31165-71). Membranes were removedby centrifugation at 130000 g for 40 minutes. Part of the supernatantcontaining the cytoplasmic fraction with approximately 1600 mg proteinwas used for the further purification. Ammonium sulfate (0.4 M) wasadded to the cytoplasmic fraction. Half of this sample was loaded onto aPhenyl-Sepharose high performance column (1.6 cm×10 cm) equilibratedwith buffer A (25 mM Tris/HC1, 20 mM MgSO₄, 0.4 M (NH₄)₂SO₄, 20%glycerol, pH 7.5). Methylviologen-dependent formate dehydrogenaseactivity elutet around 0.33 M (NH₄)₂SO₄ in a linear gradient of 120 mlfrom 0.4 M to 0 M (NH₄)₂SO₄. This step was repeated with the second halfof the sample in a separate run to gain more protein since otherwiselarge amounts of the activity eluted in the flowthrough. The pooledfractions of both runs were diluted to a conductivity of below 10 mS/cmwith buffer C (25 mM Tris/HC1, 20 mM MgSO₄, 20% glycerol, pH 7.5) andapplied to a Q-Sepharose high performance column (2.6 cm×5 cm)equilibrated with buffer C. Protein was eluted with a linear gradient of160 ml from 150 mM to 500 mM NaCl. Formate dehydrogenase eluted ataround 360 mM NaCl. Pooled fractions were concentrated usingultrafiltration in 100-kDa VIASPIN tubes and applied to a Superose 610/300 GL prepacked column equilibrated with buffer C and eluted at aflow rate of 0.5 ml/min. Formate dehydrogenase activity eluted as asingle peak. Pooled fractions were stored at 4° C.

Measurements of HCDR activity were performed at 30° C. with in buffer 1(100 mM HEPES/NaOH, 2 mM DTE, pH 7.0) in 1.8 ml anaerobic cuvettessealed by rubber stoppers, containing 1 ml buffer and a gas phase of0.8×10⁵ Pa H₂ and 0.2×10⁵ CO₂. Production of formate was measured usingFormate dehydrogenase of Candida boidinii with 2 mM NAD in the assay andproduction of NADH was followed.

For measurements with ferredoxin as electron carrier Ferredoxin waspurified from Clostridium pasteurianum. For reduction of ferredoxin COdehydrogenase of A. woodii was purified and in these experiments the gasphase of the cuvettes was changed to 100% CO (1.1×10⁵ Pa).

Whole Cells

For experiments with whole cells, the inventors used cell suspensions ofA. woodii for the conversion of H₂ and CO₂ to formate. The energymetabolism of A. woodii is strictly sodium ion dependent, and the ATPsynthase uses Na⁺ as the coupling ion. Thus, by omitting sodium ions inthe buffer or by adding sodium ionophores (the inventors used theionophore ETH2120 in this study), it is possible to switch off theenergy metabolism specifically. Cells suspended in imidazole buffer (50mM imidazole, 20 mM MgSO₄, 20 mM KCl, 4 mM DTE, pH 7.0) containing 20 mMNaCl converted H₂+CO₂ to acetate, and only small amounts of formate wereproduced from a gas phase of 0.8×10⁵ Pa H₂ and 0.2×10⁵ CO₂. By addingETH2120 (30 μM), acetate production ceased almost completely and formatewas produced with an initial rate of 2 μmol/min×mg cell protein (FIG.1). In agreement with the results obtained from the purified enzyme,formate production was also observed when hydrogen was absent and theelectron donor was CO but with lower rates compared to hydrogen aselectron donor.

The results in FIG. 1 demonstrate that a maximal amount of around 8 mMof formate was produced in this experiment. The inventors next tested,if the final formate concentration is proportional to the initial gaspressure. From 0.5 to 2×10⁵ Pa H₂+CO₂ the final formate concentrationdid not increase. Since the inventors observed a pH drop during theexperiment, the inventors examined if the lower pH is the limitingfactor. Increasing the buffer concentration from 50 to 200 mM resultedin a final formate concentration of around 14±3 mM. If CO₂ was exchangedwith KHCO₃ the effect was even more dramatic. By using the base HCO₃ ⁻the overall process is almost pH neutral compared to the production offormic acid from CO₂. The genome of A. woodii encodes for acarboanhydrase that allows the rapid interconversion of CO₂ and HCO₃ ⁻.FIG. 2 shows the production of formate from initially 300 mM KHCO₃ (with1×10⁵ Pa H₂) compared to CO₂ as substrate. Finally up to 184±5 mMformate were produced with KHCO₃.

The relationship of the final formate concentration to the initialconcentration of HCO₃ ⁻ is shown in FIG. 3. Up to 300 mM HCO₃ ⁻, thefinal formate concentration increases with increasing substrateconcentration. Furthermore the final formate concentration fits well tothe theoretic thermodynamic limit of the reaction underlining theindependence of the carboxylation of CO₂/HCO₃ ⁻ from other cellularprocesses. At 1×10⁵ Pa H₂, the thermodynamic equilibrium isapproximately [HCO₃ ⁻]=[HCOOH], so equimolar concentrations of substrateand product. At concentrations of HCO₃ ⁻ above 300 mM, this relationshipdoes not exist anymore and the final amount of formate produced ceasedaround 300 mM.

1. A method for storing gaseous hydrogen, comprising the steps ofproducing methanoate (formate) through contacting gaseous hydrogen withcarbon dioxide in the presence of a hydrogen dependent carbon dioxidereductase (HDCR), and thereby storing of said gaseous hydrogen.
 2. Themethod according to claim 1, wherein said HDCR is selected from FdhF1 orFdhF2 of Acetobacterium woodii.
 3. The method according to claim 1,wherein said HDCR is part of an enzyme complex with a formatedehydrogenase accessory protein, an electron transfer protein, or asubunit harboring the active site characteristic of an[FeFe]-hydrogenase.
 4. The method according to claim 1, wherein saidmethod is performed under standard ambient temperature and pressure orat between about 20° C. to about 40° C. and normal pressure.
 5. Themethod according to claim 1, wherein said method further comprises aninhibition of the cellular metabolism to further metabolize formate. 6.The method according to claim 1, further comprising the step ofconverting carbon monoxide into carbon dioxide using a CO dehydrogenaseand a ferredoxin.
 7. The method according to claim 1, wherein saidmethod is performed in vitro, in vivo and/or in culture.
 8. The methodaccording to claim 1, further comprising the release of hydrogen fromthe methanoate as produced.
 9. A method for decontaminatingCO-contaminated or polluted gases, comprising performing a methodaccording to claim 1 using said CO-contaminated or polluted gas as asubstrate.
 10. The method according to claim 9, wherein saidCO-contaminated or polluted gas is synthesis gas.
 11. A recombinantbacterial organism comprising a genetic modification, wherein saidgenetic modification comprises transformation of said microorganism withexogenous bacterial nucleic acid molecules encoding the proteins FdhF1and/or FdhF2, FdhD, HycB1 and/or HycB2, and HydA2, HycB3, and optionallyAcsA of Acetobacterium woodii, or homologs thereof, whereby expressionof said proteins increases the efficiency of producing formate from CO₂,and/or CO and H₂.
 12. A method for storing gaseous hydrogen, comprisingthe steps of producing methanoate (formate) through contacting gaseoushydrogen with carbon dioxide in the presence of a hydrogen dependentcarbon dioxide reductase HDCR and thereby storing of said gaseoushydrogen wherein said method comprises the use of the recombinantbacterial organism according to claim
 11. 13. (canceled)
 14. The methodaccording to claim 3, wherein said formate dehydrogenase accessoryprotein is FdhD of Acetobacterium woodii or a homolog thereof. 15 Themethod according to claim 3, wherein said complex further comprises a COdehydrogenase and a ferredoxin, or a homolog thereof.
 16. The methodaccording to claim 15, wherein said CO dehydrogenase is AcsA ofAcetobacterium woodii, or a homolog thereof.
 17. The method according toclaim 3, wherein said electron transfer protein is HycB1 or HycB2 ofAcetobacterium woodii, or a homolog thereof.
 18. The method according toclaim 3, wherein said subunit harboring the active site characteristicof an [FeFe]-hydrogenase is HydA2 of Acetobacterium woodii, or a homologthereof.
 19. The method according to claim 5, wherein said inhibition ofcellular metabolism is accomplished by Na depletion using sodiumionopheres.